Enzyme products

ABSTRACT

THIS INVENTION RELATES TO PARTICULATE ENZYME PRODUCTS AND TO PROCESSES FOR PREPARING THE SAME; THESE PRODUCTS CONTAIN ENZYMES PRODUCED BY BACILLUS SUBTILLIS, HAVE A BULK DENSITY OF FROM ABOUT 0.25 GRAM/CC. TO ABOUT 2.0 GRAMS/CC. AND HAVE A CRITICAL PARTICLE SIZE.

United States Patent M 3,573,170 ENZYME PRODUCTS Sidney G. Clark and Harold E. Feierstein, St. Louis, Mo., assignors to Monsanto Company, St. Louis, M0. N0 Drawing. Filed Sept. 19, 1968, Ser. No. 760,981 Int. Cl. (107g 7/02 US. Cl. 195-63 12 Claims ABSTRACT OF THE DISCLOSURE This invention relates to particulate enzyme products and to processes for preparing the same; these products contain enzymes produced by Bacillus subtilis, have a bulk density of from about 0.25 gram/cc. to about 2.0 grams/ cc. and have a critical particle size.

This invention relates to novel compositions of matter and to a process for preparing the novel compositions of matter. More particularly, this invention relates to particulate enzyme products, to process for preparing said enzyme products and to detergent compositions including the same.

Enzymes are used in laundry compositions to remove stubborn stains such as those caused by proteins, carbohydrates, fats and the like. It has been found that enzymes produced by Bacillus subtilis micro-organisms are very effective in removing these stains. Consequently, it would be very desirable to include enzymes produced by Bacillus subtilis in detergent compositions, particularly in white compositions which are very attractive to the consumer. Some housewives prefer a white detergent because they believe it will get their clothes much cleaner than an off white or gray detergent. Generally, this is not the case as color usually has no bearing on detergency.

Enzymes produced by Bacillus subtilis micro-organisms must be purified by separating them from material used in their production. This separation step is very complex and time consuming. Although 100% pure enzyme can be recovered, this is generally not done because of cost considerations and decreased yields. The enzyme material that is recovered in good yields, however, has a tan color. This is especially true when appreciable amounts of enzyme material are prepared, for inclusion in detergents; moreover, when used in white detergents, this enzyme material tends to darken these laundry compositions giving them an off white or gray appearance. Attempts to remove these color bodies by further processing have not been successful. It is believed, therefore, that enzyme materials having a tan color which can be incorporated into a white detergent composition without visibly changing the color of these compositions are an advancement in the art.

In accordance with this invention, it has been found that a particulate enzyme product having a particle size so that 100% of the particles pass through a No. 40 mesh U.S. standard screen, 95% of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 170 mesh U.S. standard screen, and 95 of the particles are retained on a No. 140 mesh U.S. standard screen and having a bulk density of about 0.25 gram/ cc. to about 2.0 grams/ cc. can be included in a white detergent composition without a visible color change. A novel process for producing the above mentioned product involves the subjection of enzyme material to pressure, to increase the density of said material, the formation of thin sheets or pellets of said material, and reduction of said sheet or pellets to the desired size. By increasing the density, a product results that nearly matches the densities of the commercially available detergent compositions, and, therefore, the products of the instant invention can be 3,573,170 Patented Mar. 30, 1971 readily incorporated into these detergent compositions with little or no segregation.

The enzymes that are used in accordance with this invention are produced by Bacillus subtilis. The term Bacillus subtilis" as used herein also refers to mutants of said organisms. Generally, Bacillus subtilis produces a mixture of enzymes; a neutral protease (maximum activity at a pH of 7 to 7.5) an alkaline protease (maximum activity at a pH of 10 to 11) and amylase. The enzymes are produced during fermentation of Bacillus subtilis. Various processes for fermentation of Bacillus subtilis may be used such as those described in Kirk and Othrner, Encyclopedia of Chemical Technology (2nd ed.), VIII, p. 173.

The above mentioned enzymes must be separated from other materials used in their production before employing them in the present invention. As mentioned before, 100% pure enzyme (free of an inert diluent) could be recovered, however, this is not generally done because of cost considerations and decreased yields. In fact, an enzyme material having a tan color employed in this invention contains about 70% to about 99.5% of an inert diluent hereinafter to be discussed. Accordingly, the enzyme material used in this invention contains from about 1% to about 99.9% by weight of an inert diluent and preferably from about 70% to about 99.5% by weight based on the weight of the enzyme materials.

Various tests can be used to determine the enzyme activity. Protease activity is determined by either the Milk Clot Method or the Casein Method. The latter method is described in the Journal of General Physiology, XXX (1947), p. 291, and Methods of Enzymology (New York: Academic Press, 1955), II, p. 80. According to this test, a protease catalyzes the hydrolysis of casein for a definite period of time at a given temperature and pH. This reaction is then stopped by the addition of trichloroacetic acid and this solution filtered. The color of the filtrate is developed by Folin reagent and the level of enzyme activity is measured spectrophotometrically. The amylase activity can be determined by various tests, for example, the test described in Methods of Enzymology (New York: Academic Press, 1955), II, p. 149.. According to this test, amylase catalyzes the hydrolysis of the starch to reduce sugar at a given time and temperature. The reaction is stopped and color developed by the addition of dinitrosalicylic acid. The optical density of the solution is estimated from a standard curve prepared with known amounts of maltase hydrate.

Preferred enzymes utilized in accordance with this invention are produced by a mutated Bacillus subtilis microorganism. A description and method for cultivating the organism is disclosed in US. Patent No. 3,031,380. A culture of this organism has been deposited with the United States Department of Agriculture, Agricultural Research Service, Northern Utilization Research and Developrnent Division, 1815 North University Street, Peoria, Ill. 61604 and has been assigned NRRL No. B- 3411. After fermentation of this mutated Bacillus Subtilis organism and subsequent separating, an enzyme material is recovered which contains a neutral protease, alkaline protease and amylase and about 70% to about by weight based on the weight of the enzyme material of an inert diluent. The neutral protease activity (pH 7 to 7.5) of this material is about 700,000 to about 1.2 million units per gram of enzyme material and the alkaline protease activity is about 250,000 to about 400,000 units per gram of this material as determined by the Casein Method. The amylase activity of this material is about 300,000 to about 350,000 units per gram of material as determined by the Starch Assay Method.

By subjecting enzyme material recovered from Bacillus subtilis organisms to various separating techniques, a

material containing 1 or different combinations of enzymes can be obtained. For example, the alkaline protease in material recovered from Bacillus subtilis No. B3411 herementioned can be inactivated by the use of diisopropyl fluorophosphate producing a material containing neutral protease and amylase or the neutral protease can be inactivated by the use of chelating agents such as ethylenediamenetetracetic acid, giving a material containing alkaline protease and amylase. By using an exchange resin essentially pure neutral protease can be separated from this enzyme material.

By variations of fermentation processes for micro-organisms, enzyme mixtures having different activities can be obtained. For example, copending U.S. application Ser. No. 752,453, filed Aug. 14, 1968, entitled Fermentation Process for Enzyme Production, and assigned to the same assignee as the instant application, discloses fermentation processes for Bacillus subtz'lis No. B-34l1 in which mixtures of enzymes having increased alkaline protease activity are produced.

As mentioned before, an inert diluent in an amount of from about 1 to about 99.9% and preferably from about 70% to about 99.5% is present. The term inert diluent refers to those materials that are inert with respect to the enzyme and includes materials remaining with the enzyme after separation (processing diluent), materials added to further reduce the activity of the enzyme ma terial or both. Enzyme activity is generally reduced, as only a small amount is needed in a detergent composition to obtain improved performance. Examples of the former include nutrients, by-products, and waste products. Examples of the latter include, for example, inorganic salts, organic salts, carbohydrates, mineral fibers, resins, hydrocarbons and the like. Illustrative inorganic salts include sodium bicarbonate, sodium sulfate, magnesium sulfate, borax, calcium sulfate, aluminum oxide, aluminum silicate, calcium oxide, and the like. Other salts include the trialkali metal phosphates such as trisodium phosphate, dipotassium phosphate and dialkali metal hydrogen phosphate, such as disodium hydrogen phosphate and dipotassium hydrogen phosphate. Still other alkali metal phosphates such as tetrapotassium pyrophosphate, tetrasodium pyrophosphate are used as well as the alkali metal tripolyphosphates such as sodium tripolyphosphate, or the alkali metal phosphates such as sodium hexametaphosphate. Examples of organic salts include aluminum tristereate, calcium laureate, lithium laureate. Illustrative carbohydrates include carboxymethylcellulose and sucrose. Illustrative mineral fibers include asbestos. Examples of resins include the salts of olefin maleic anhydride and a substituted olefin maleic anhydride including the alkali metal, ammonium and amine salts of ethylene maleic anhydride. The following also may be used, the amino carboxylic acids and salts such as sodium potassium and ammonium salts of amino trimethylene phosphonic acid as well as the free acid in the diphosphonic acid and salt. Methylene diphosphonic acid and 1-hydroxy-1,1-ethylidene diphosphonic acid other diluents such as soda ash, diatomaceous earth, silica powdered talc, kaolinate clay and the like can be used. Other illustrative compounds include the alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; the alkane sulfates, such as sodium dodecyl sulfate; and the sulfated oxyethylated phenols, such as sodium tetradecyl phenoxy triethyleneoxy sulfates. Still further examples of compounds are usually made by reacting an alkylene oxide such as ethylene oxide, butylene oxide, propylene oxide and the like with fatty acids, the straight or branched chain alcohols, phenols, thiophenols, amides, and amines to form polyoxyalkylene glycol ethers and esters, polyoxyalkylene alkyl phenol and polyoxyal kylene thiophenols, and polyoxyalkylene amides and the like. Other compounds can be broadly described as compounds which have both an anionic and cationic group in their structure. Illustrative compounds are the amido alkane sulfonates, such as sodium C-tridecyl, N-methyl,

amido ethyl sulfonate. Also mixtures of any of the above diluents can be employed in this invention. Especially preferred materials include sodium tripolyphosphate, sodium silicate, sodium borate, sodium sulfate, borax and starch. Generally, any of the above mentioned materials will be white so they can be admixed into white detergent compositions.

The amount of materials used to reduce enzyme activity is generally from about 11% by weight to about 99% by weight based on the weight of the inert diluent. For example, a 50% reduction in enzyme activity of a material containing about 70% by weight of a processing inert diluent is accomplished by using 50% sodium tripolyphosphate and 50% of the enzyme material resulting in an enzyme material having about 85% by Weight of an inert diluent, about 59% by weight of the inert diluent being sodium tripolyphosphate and about 41% by weight of the inert diluent being a processing inert diluent. Another illustration wherein an reduction in enzyme activity of an enzyme material containing about 80% by weight of a processing inert diluent is carried out by adding about 80% sodium sulfate and about 20% of the enzyme material resulting in an enzyme material having about 96% by weight of an inert diluent about 84% by weight of the inert diluent being sodium sulfate and about 16% by weight being a processing inert diluent. A still further example wherein a 20% reduction in enzyme activity of an enzyme material containing about 85% by weight of processing inert diluent is accomplished by using about 20% starch and 80% of the enzyme material resulting in an enzyme material having 88% by weight of an inert diluent about 23% by weight of the inert diluent being starch and about 77% by weight of the inert diluent being a processing diluent.

Still another illustration wherein a 98.5% reduction in enzyme activity of a material containing 80% by weight of a processing inert diluent is accomplished by using 15% alkyl benzene sulfonate, 20% sodium sulfate, 10% sodium silicate, 53.5% sodium tripolyphosphate and 1.5% of the enzyme material resulting in an enzyme material having about 99.7% inert diluent, about 1.2% by weight of the inert diluent being a processing diluent and 98.8% by weight being the above mentioned ingredients. It will readily be seen that by using the stated quantities of the above mentioned materials in the last illustration, that a detergent composition may be prepared having the amount of enzyme material generally used in detergent compositions.

As can readily be appreciated, an enzyme material having any desired enzyme activity can be formulated and used in the novel process of this invention hereinafter to be described.

Generally, the particle size of the above mentioned enzyme materials in the absence of any materials used to reduce enzyme activity, is generally from about 12 to about 15 microns. These particles have a tan color, are dusty and difiicult to handle. By subjecting these materials to the novel process hereinafter to be described, a particulate enzyme product having a particle size so that 100% of the particles pass through a No. 40 mesh U.S. standard screen, of the particles pass through a No. 50 mesh U.S. standard screen, of the particles are retained on a No. 170 mesh U.S. standard screen, and 95% of the particles retained on a No. mesh U.S. standard screen is obtained. A product having this particle size can be incorporated into a detergent composition having a white appearance and there is no visible color change in the detergent composition. The material is also easier to handle. The particle size is very critical to this invention, for example, an enzyme product having a particle size such that 100% of the particles pass through a No. 30 mesh U.S. standard screen, 90% of the particles pass through a No. 40 mesh U.S. standard screen, 80% Of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No.

170 mesh U.S. standard screen and 95% of the particles are retained on a No. 140 mesh U.S. standard screen will show up as tan particles in a white detergent composition or another product having a particle size such that 100% of the particles pass through a No. 40 mesh U.S. standard screen, 95% of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 250 mesh U.S. standard screen, 90% of the particles are retained on a No. 170 mesh U.S. standard screen and 80% of the particles are retained on a No. 140 mesh U.S. standard screen will tend to darken a white detergent composition giving it a gray appearance.

The density of the above mentioned enzyme materials, in the absence of any materials utilized to reduce enzyme activity is generally about .23 gram/cc. By utilizing the novel process of this invention, a product can be prepared having density from about 0.25 gram/cc. to about 2 grams/cc. and preferably from about 0.3 gram/cc. to about 1.0 gram/ cc. It will readily be appreciated that an enzyme product having a density Within the above mentloned range, can readily be admixed with the commercially available detergents which have bulk densities Within a range from about 0.3 gram/cc. to about 1.2 grams/ cc.

The novel process of this invention involves applying pressure to the enzyme material thereafter grinding it to the critical particle size hereinbefore mentioned. The amount of pressure used in accordance with the novel process is from about lb./sq. in. to about 600 lb./sq. in. and preferably from about 25 lb./sq. in. to about 100 lb./sq. in.

Any suitable machinery can be used to carry out the novel process of this invention, for example, compressing machines, presses, hydraulic presses, or automatic compressing machines can be used to apply pressure. In order to break up the particles, any of the standard mills are suitable, such as direct pressure mills, disc mills, roller mills, hammer mills, ball mills and the like. The enzyme product can be screened to the proper size by using a stationary screen, a moving screen, a vibrating screen, or a shaking screen. An especially preferable device is a compression machine employing compression rollers. The rollers are so arranged to form the enzyme material into a sheet which is approximately to A3 thick, this sheet is brittle and can be easily fractured into smaller segments which are then screened to the critical size as mentioned hereinbefore.

As mentioned before, detergent formulations comprising an active, builder and an enzyme product produced according to this invention have a white appearance and therefore are very attractive to the housewife. The amount of enzyme product used is generally from about .01% to about 10% by weight and preferably from about 0.1% to about 3% by weight based on the weight of the detergent composition. In terms of total protease activity, both alkaline and neutral, this is about 160 units per gram to about 160,000 units per gram and preferably from about 1600 units to about 48,000 units per gram.

The amount of active and builder is generally from about 90% to about 99.99% and preferably from about 97% to about 99.9% by Weight based on the weight of the detergent composition.

Generally, the surface active agents which are employed in the detergent compositions are various soaps such as those produced from the saponification of a fatty acid such as palmetic oleic, and the snythetic organic surfactant including the anionic and nonionic and arnpholytic types and mixtures thereof. Anionic synthetic surface active agents are generally described as those compounds which contain hydrophilic and lyophilic groups in their molecular structure and ionize an aqueous medium to give anions containing both the lyophilic groups and the hydrophilic group. The alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; the al kane sulfates, such as sodium dodecyl sulfate; and the sulfates oxyethylated phenols, such as sodium tetradecyl phenoxy triethyleneoxy sulfate, are illustrative of the well-known class of anionic type of surface active compounds.

Nonionic surface active compounds can be broadly described as compounds which do not ionize but acquire hydrophilic characteristics from an oxygenated side chain such as polyoxyethylene and the lyophilic part of the molecule may come from fatty acids, phenol, alcohols, amides, or amines. The compounds are usually made by reacting an alkylene oxide such as ethylene oxide, butylene oxide, propylene oxide and the like with fatty acids, the straight or branched chain alcohols, phenols, thiophenols, amides, and amines to form polyoxyalkylene glycol ethers and esters, polyoxyalkylene alkyl phenol and polyoxyalkylene thiophenols, and polyoxyalkylene amides and the like. It is generally preferred to react from about 1 to about 30 moles of alkylene oxide per mole of the fatty acids, alcohols, phenols, thiophenols, amides or amines. Illustrative of the surface active agents include the product obtained from condensing ethylene oxide with the following: propylene glycol, ethylene diamine, diethylene glycol, dodecyl phenol, nonyl phenol and the like.

Amphoteric surface active compounds can be broadly described as compounds which have both an anionic and cationic group in their structure. Illustrative of the amphoteric surface active agents are the amido alkane sulfonates, such as sodium C-tridecyl, N-methyl, amido ethyl sulfonate.

Other individual compounds which are illustrative of the foregoing classes of surface active agents are wellknown in the art and can be found in standard detergent reference materials such a Surface Active Agents, Schwartz and Perry, Interscience Publishers, Inc., New York, NY. (1949).

The amount of active used in practicing this invention is such that the active to builder ratio is from 1:10 to about 10:1 and preferably from about 1:1 to about 1:7.

Builders which can be employed in detergents in ac cordance with this invention include various organic and inorganic salts and mixtures thereof. The amount of builder used in practicing this invention is such that the active to builder ratio is from about 1:10 to about 10:1 and preferably from about 1:1 to about 1:7.

The organic compounds which are used include aminopolycarboxylic acids, their water soluble salts, such as nitrilotriacetic acid and its alkali metal salts; the amino tri(lower alkylidenephosphonic acid) and their water-soluble salts such as amino tri(methylenephosphonic acid) and its alkali metal salts and the alkylene diphosphonic acids and their water-soluble salts such as methylene diphosphonic acid and its alkali metal salts and mixtures of the foregoing compounds.

Additionally in many of the detergent compositions other additives are present such as anti-redeposition agents, brightening agents, corrosion inhibitors, perfumes, inert filler, blueing agents and the like. Typical examples of such additives are sodium carboxymethyl cellulose, polyvinyl alcohol, sodium sulfate, sodium silicate, methyl cellulose and sodium carbonate.

The following nonlimiting examples illustrate the present invention. All parts, portions and percentages are by weight unless otherwise stated.

EXAMPLE Into a container is placed 100 parts of enzyme material recovered from a fermentation of Bacillus subtilis No. B-3411 containing about of an inert processing diluent. The neutral protease activity (pH 7) is about 1.1 million units per gram and the alkaline protease activity (pH is about 290,000 units per gram as determined by the Casein Method. The amylase activity is about 300,000 units per gram as determined by the Starch Assay Method. The enzyme material has a tan color. The enzyme material is moved to another holding tank by means of a conveyor. The enzyme material is fed at the rate of 500 lbs/hr. to two 10" compression rollers.

Roller pressure is about 25 p.s.i.g. The rollers compress the enzyme material and it emerges from the rollers in an irregular brittle sheet, measuring about 3" x 5 x /8". This sheet is then fractured into smaller particles by a hammer mill. These particles are screened so that 100% of the particles pass through a No. 40 mesh U.S. standard screen, 95% on the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 170 mesh U.S. standard screen and 95% of the particles are retained on a N0. 140 mesh U.S. standard screen. An analysis showed this enzyme product (product A) to have a bulk density of 0.4 gram/cc.

A similar enzyme product (product B) as above is prepared having the following particle size, 100% of the particles pass through a No. 30 mesh U .8. standard screen, 90% of the particles pass through a No. 40 mesh U.S. standard screen, 80% of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 170 mesh U.S. standard screen and 95% of the particles are retained on a No. 140 mesh U.S. standard screen.

Another enzyme product (product C) similar to the above is prepared having the following size 100% of the particles pass through a No. 40 mesh U.S. standard screen, 95% of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 250 mesh U.S. standard screen, 90% of the particles are retained on a No. 170 mesh U.S. standard screen and 80% of the particles are retained on a No. 140 mesh U.S. standard screen.

In order to compare the color of a White detergent composition containing enzyme products, the following test is conducted. 0.5 part of enzyme product A is admixed with 99.5 parts of a white detergent composition having the following formula:

Percent Sodium dodecyl benzene sulfonate Sodium tripolyphosphate 45 Sodium silicate 10 Sodium sulfate Sodium carboxymethylcellulose 1 Water 9 Two other detergent compositions are prepared in the same manner except 0.5 part of enzyme product B is substituted for product A in one, and 0.5 part of product C is substituted for product A in the other. A detergent composition without an enzyme product is used as a control.

A visual examination is made of these compositions. The color of the composition containing product A is unchanged. Tan speckles are seen in the composition containing product B. The composition containing product C has a definite tan cast.

In another test, the reflectance, of a 1% blend of each of the above detergent compositions containing the enzyme products is determined using 21 Gardner Color Difference Meter. The results are given in Table 1 below.

TABLE 1 Detergent composition plus: Gardner reading No enzyme product 77.6 Product A 77.5 Product B 73.4 Product C 72.1

A difference of less than 1.0 unit cannot be seen by the eye. Here, Product A differs by only 0.3 unit and therefore is not perceptible while the other products having particle sizes outside the narrow range, differing by 1.7 to 2.8 units, can be seen in the detergent composition.

EXAMPLE II Into a container is placed 20 parts enzyme material having the same activity and inert diluent as in Example I, parts of sodium tripolyphosphate and blended. The enezyme material is processed in the same manner as Example I giving Product A having 100% of the particles passing through a No. 40 mesh U.S. standard screen, of the particles passing through a No. 50 mesh U.S. standard screen, of the particles retained on a No. 170 mesh U.S. standard screen and 95 of the particles retained on a No. mesh U.S. standard screen. The enzyme product has a tan color.

Similar products B, C and D are prepared by substituting 80 parts of sodium sulfate, 80 parts of borax and 80 parts of starch respectively, for 80 parts of sodium tripolyphosphate.

In order to test the eifect of the enzyme products on the color of a white detergent composition, 10 parts of enzyme Product A is admixed with 90 parts of a white detergent composition having the same formulation as that used in Example 1. Other detergent compositions are prepared in exactly the same manner, except Product B replaces A in one, Product C replaces A in another and Product D replaces A in still another. One detergent composition is used as a control.

A visual examination is made, which shows no color change in any of the compositions.

The reflectance of a 1% blend of each of the products is determined by using a Gardner Color Difference Meter. The results are given below.

TABLE 2 Detergent compositions plus: Gardner reading No enzyme product 87.2 Enzyme Product A 87.0 Enzyme Product B 86.8 Enzyme Product C 86.2 Enzyme Product D 87.1

A difference of less than one reflectance unit is not visible to the eye. As long as the size specified in accordance with this invention is maintained. The detergent composition appears White as the above results indicate.

EXAMPLE III Using a conventional machine for making pellets, and maintaining the pressure at about 50 lb./sq. in. to about lb./sq. in., 100 parts of an enzyme material having about 80% inert diluent are converted to pellets. They are then ground to a size so that 100% of the particles pass through a No. 40 mesh U.S. standard screen, 95 of the particles pass through a No. 50 mesh U.S. standard screen, and 95 of the particles are retained on a No. 140 mesh U.S. standard screen.

Another product is prepared by blending 80 parts of enzyme material with 20% sodium sulfate and converting this blend to pellets which are then ground to the same size as mentioned above. Another product is produced in the same manner as mentioned above, with the exception that the enzyme material is blended with sodium tripolyphosphate. Another example is prepared with the exception that the enzyme material is blended with sodium borax. Each of these enzyme products is added to a white detergent composition with the same desirable results.

The enzyme product of the present invention possesses many advantageous properties. The particle size enables the tan colored enzyme product to be admixed with a white detergent composition without visibly changing the white appearance. The particle size improves the rate of solution in water of the product. The density is such that segregation in detergent compositions is cut down or eliminated. Due to these properties, it will readily be appreciated that these enzyme products can be added to detergent compositions, particularly to attractive white detergent compositions.

From a consideration of the above specification, it will be understood that many improvements and modifications in the details may be made without departing from the spirit and scope of the invention. It is to be understood, therefore, that the invention is not limited except as defined by the appended claims.

What is claimed is:

1. A particulate enzyme product comprising enzymes produced by Bacillus subtilis, said product having a bulk density of from about 0.25 to about 2.0 grams/cc. and a particle size such that 100% of the particles of said product pass through a No. 40 mesh U.S. standard screen, 95% of the particles pass through a No. 50 mesh U.S. standard screen, 100% of the particles are retained on a No. 170 mesh U.S. standard screen, and 95% of the particles are retained on a No. 140 mesh U.S. standard screen, said product containing from about 1 to about 99.9 by weight of an inert diluent.

2. A product according to claim 1 wherein said enzymes are produced by a mutated Bacillus subtilis NRRL No. B-3411.

3. A product according to claim 1 wherein said product contains from about 70 to about 99.5% by weight of an inert diluent.

4. A product according to claim 1 wherein about 11% to about 99% by weight of said inert diluent is an inorganic salt.

5. A product according to claim 4 wherein the inorganic salt is selected from the group consisting of borax, soidum sulfate and disodium phosphate.

6. A product according to claim 5 wherein said enzymes are produced by a mutated Bacillus subtilis NRRL No. B-3411.

7. A product according to claim 3 wherein said inert diluent is a processing diluent.

8. A product according to claim 7 wherein said enzymes are produced by a mutated. Bacillus subtilis NRRL No. 13-3411.

9. A process for the production of an enzyme product containing enzymes which comprises increasing the density of the enzyme material by applying pressure, said pressure being from about 10 lb./sq. in. to about 600 1b./sq. in and thereafter grinding said enzyme materials to a particle size such that 100% of the particles pass through a No. mesh U.Si. standard screen, of the particles pass through a No. 50 mesh U.S. standard screen, of the particles are retained on a No. 170 mesh U.S. standard screen, and 95 of the particles are retained on a No. mesh U.S. standard screen whereby a particulate enzyme product having a bulk density of from about 0.25 to about 2.0 grams/cc. is obtained.

10. A process according to claim 9 wherein said enzymes are produced by a mutated Bacillus subtilis NRRL No. B-34l1.

11. A process according to claim 9 wherein said pressure is applied by means of rollers, said rollers are used to form a sheet of material having a thickness of about to about A3".

12. A process according to claim 11 wherein said enzymes are produced by a mutated Bacillus subtilis NRRL No. 3-3411.

References Cited UNITED STATES PATENTS 3,451,935 6/1969 Roald et al. 19568X LIONEL M. SHAPIRO, Primary Examiner U.S. Cl. XR. 

